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123). In this study, Martinez-Contreras and her associates report the results of recent research that has provided additional evidence concerning the function of these proteins in precursor-messenger RNA (pre-mRNA) splicing (2007).
The splicing repression can function in two discrete ways in heterogeneous nuclear RNP proteins; the first way is by antagonizing the recognition of splice sites directly and the second way is through interference with the binding of proteins that are bound to enhancers (Martinez-Contreras et al., 2007). A growing body of research concerning the role of hnRNP proteins has determined that these proteins can restrict communication between factors bound to different splice sites; by contrast, a number of studies have identified a positive role for some hnRNP proteins in pre-mRNA splicing (Martinez-Contreras et al., 2007). Moreover, the research to date suggests cooperative interactions between bound hnRNP proteins that may facilitate splicing between specific pairs of splice sites while concomitantly repressing other combinations (Martinez-Contreras et al., 2007). Therefore, it has become increasingly apparent that hnRNP proteins employ a wide range of methods to control splice site selection in a fashion that is important for alternative as well as constitutive pre-mRNA splicing (Martinez-Contreras et al., 2007).
The functioning of hnRNPs has also been investigated by Prahl, Vilborg, Palmberg, Jornvall, Asker and Wiman (2008) who report that hnRNPs take part in the maintenance of telomere length, transcriptional regulation, alternative pre-mRNA splicing and pre-mRNA 30 end processing within the nucleus. According to these researchers, "In the cytoplasm, hnRNPs can regulate mRNA localization, translation and turnover. hnRNP A2 (36 kDa) and B1 (38 kDa) are isoforms derived from the same gene and differ by only 12 amino acids, due to the presence of exon 2 in the B1 transcript" (Prahl et al., 2008, p. 2173). In transformed cells, the targeting of hnRNP A2/B1 facilitates the death of cells; however, targeting hnRNP A2/B1 in primary cells does not promote their death (Prahl et al., 2008). In addition, the suppression of hnRNP A2 results in a non-apoptotic inhibition of cell proliferation (Prahl et al. 2008).
A study by David, Chen, Assanah, Canoll and Manley (2010) notes that there are three heterogeneous nuclear ribonucleoprotein (hnRNP) proteins: (a) polypyrimidine tract binding protein (PTB, also known as hnRNPI); (b) hnRNPA1 and (c) hnRNPA2; all three of these hnRNP proteins repressively bind to sequences that are adjacent to exon 9 causing exon 10 inclusion. In their study, these researchers also demonstrate that the oncogenic transcription factor c-Myc upregulates the transcription of hnRNPA1, hnRNPA2 as well as PTB thereby assuring an elevated ratio for PKM2/PKM1 (David et al., 2010). Establishing a relevance to the function of these proteins with regards to cancer, these researchers also determined that the overexpression of human gliomas PTB, hnRNPA1, c-Myc, and hnRNPA2 are congruent with the same type of overexpression that occurs in PKM2 (David et al., 2010). Based on these results, these researchers concluded that these findings define a pathway that regulates an alternative splicing event that is implicated in tumor cell proliferation (David et al., 2010).
The role of PKM2 proteins in cancer proliferation was also the subject of a study by Clower, Chatterjee, Wang, Cantley, Vander Heiden and Krainer (2010), who report, "Cancer cells exhibit a metabolic phenotype characterized by increased glycolysis with lactate generation, regardless of oxygen availability -- a phenomenon termed the Warburg effect" (p. 1894). In addition, Clower et al. point out that recent research has shown that the expression of the type II isoform of the pyruvate-kinase-M gene (PKM2, referred to as PK-M by these researchers) is "a critical determinant of this metabolic phenotype, and confers a selective proliferative advantage to tumor cells in vivo" (2010, p. 1894). The significance of this finding is summarized by these researchers thusly: "This finding adds to the growing body of evidence that alterations in alternative pre-mRNA splicing play important roles in different aspects of cancer progression" (Clower et al., 2010), p. 1894).
Clearly, the expression of pre-mRNA plays an important role in the cancer-development process, and measuring the constituent elements of the process may help researchers develop superior early detection mechanisms that can provide earlier treatment and improved clinical outcomes. For instance, Clower et al. add that, "Proliferating cells and cancer cells preferentially express PK-M2 over PK-M1 at the protein level" (2010, p. 1895). Taken together, the foregoing suggests that additional research into hnRNP proteins is warranted, and the research objective of this study is discussed further below.
Work in our laboratory has previously shown that all vertebrate cells have very high concentrations of a protein called hnRNP C. In their nuclei. The function of this protein has remained elusive. It has been proposed to be involved in many cellular activities. The research objective of this study was to investigate the expression of hRaly and hnRNP C, which have been found to be ubiquitous in all tissues examined in previous studies. To this end, the study collected 24 paired tissue samples from normal and tumor cells which were then screened for hRALY and hnRNP C. expression. Another protein, though, hRaly, has been discovered that is virtually identical to hnRNP C; this protein shares fully 43% primary sequence homology with hnRNP C1 and C2. Therefore, identifying techniques that can measure RNA amounts of hnRNP C. And hRALY in both cancer and normal cells represents a timely and valuable enterprise as discussed further below.
Chapter Two: Literature Review
Background and Overview
The need for reliable biomarkers for use in the early detection of cancer has been well established, but there remains a fundamental lack of specific biomarkers that are capable of assessing the effects of long-term exposure to various environmental conditions, even over the course of several decades (Brody & Rudel, 2003). Further, women in particular at are risk for a number of diseases that may be affected by environment conditions, including breast cancer (the most common type) (Brody & Rudel, 2003), as well as uterine cancer (the fourth most common with about 40,000 new cases in the United States each year) (Identifying uterine cancer, 2006). Biomarkers may therefore help improve the assessment of various environmental exposures on the incidence of cancer in general and in women in particular (Heck, Andrew, Onega, Rigas, Jackson, Karagas & Duell, 2009). In this regard, Boukakis, Patrinou-Georgoula, Lekarakou, Valavanis and Guialis (2010) note that, The biogenesis of mRNA in higher eukaryotes is largely based on the interplay of a large number of RNA-binding proteins (RBPs). Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RBPs that are essential players in mRNA metabolism, acting as coordinators of post-transcriptional events (splicing, transport, cellular localisation, decay and translation of mRNA) by participating in an extensive network of RNA-RBP interactions" (p. 434). Likewise, according to Sun, Xu, Poon, Day and Luk (2010), early cancer detection using mRNA-level biomarkers can also help improve clinical outcomes for liver cancer patients. Similarly, Katsimpoula, Patronou-Georgoula, Makkrilia, Dimakou, Guialis, Orfandious and Syrigo (2009), the overexpression of hnRNP A2/B1 has increasingly been cited as representing a potential useful marker for early detection of lung cancer.
In addition, individual hnRNPs function in several other cellular processes are of great interest to researchers, including transcription, DNA repair, telomere biogenesis and cell signaling; consequently, any malfunctioning in the various regulatory roles gene expression in these proteins, particularly with regards to their deregulated expression in cancer, is believed to affect the physiological network of RNA-RBP interactions (Boukakis et al., 2010). Recent research in this area is discussed further below.
Recent Clinical Research
According to Sueoka, Goto, Sueoka, Kai, Kozu and Fujiki (1999), heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is an RNA binding protein and this binding protein is an essential component needed for the successful maturation of the mRNA precursor. These researchers add that, "hnRNP A2/B1 protein is a major component of the hnRNP core complex in mammalian cell nuclei. Although the function of hnRNP A2/B1 has not yet been fully elucidated, recent studies have revealed that hnRNP A2/B1 is involved in RNA splicing in nuclei as well as in mRNA transport from nucleus to cytoplasm" (Sueoka et al., 1999, p. 1404).
When hnRNP A2/B1 mRNA and hnRNP B1 mRNA were investigated separately, these researchers developed original evidence that hnRNP B1 mRNA, which is a splicing variant of hnRNP A2 mRNA, was significantly increased in lung cancer tissues compared to hnRNP A2/B1 mRNA (Sueoka et al., 1999). Further, Sueoka et al. note that the hnRNP B1-specific polyclonal antibody used in their study specifically recognized hnRNP B1 protein as a Mr. 37,000 nuclear protein through the use of Western blotting techniques; however, it did not recognize hnRNP A2 protein. As to the procedures, Sueoka et al. report that, "Immunohistochemical staining with the hnRNP B1 antibody revealed that hnRNP B1 protein was specifically stained in the nuclei of human cancer cells, and in squamous cell carcinomas in particular, but not in those of normal adjacent…[continue]
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